The goal of our research is to understand the structure and function of acetylcholine receptor-channel molecules. We propose here 6 extensions of current projects; (1) To further validate our technique for visualizing receptor-channel molecules in the post-synaptic membranes of Narcine electric tissue by lengthening them with specific ligands; to examine for the same molecules in normal and denervated muscles and in ganglia; to obtain further information about the shape and length of the molecules and their pores, and attempt to visualize channels partially plugged with specific agents during fixation; and to compare the appearance of other known and suspected channel-like molecules with that of nicotinic receptor molecules, including gap junction molecules in cardiac muscle and liver, Na ion, K ion-ATPase in dorsal electroplaque membranes, Na ions channels at nodes of Ranvier, and Ca ions channels in nerve terminals. (2) To determine which subunits of receptor molecules reach the cytoplasm by labelling the inner ends of the subunits with radioligands; and to determine which of the 4 kinds of subunits lie next to each other in the intact molecule by utilizing cleavable crosslinking agents. (3) To separate the portion of nicotinic receptor subunits which protrudes into synaptic clefts, with cleaving enzymes, isolate the tips, and determine their protein and other characteristics, and precise glycoprotein structure. (4) To complete the purification of postsynaptic membranes from the electric tissue of Narcine by an affinity technique, and determine their exact lipid composition. (5) To develop an in vitro system to study the efflux of 22Na ion from purified vesicles of postsynaptic membranes, and from reconstituted vesicles, after their exposure to physiologically relevant concentrations of acetylcholine (0.1 - 1 mM) for about 1 ms; and to examine efflux after stabilizing 1, 2 or 3 receptors on each molecule in various "resting", "irreversibly activated", and "densitized" states. (6) To attempt to incorporate receptor-channel molecules into black lipid membranes, initially by fusing unaltered postsynaptic membranes from Narcine with preformed large vesicles suitable for study by black membranes techniques.